Processes for extracting phytochemicals from pomegranate solids and compositions and methods of use thereof

ABSTRACT

Processes for producing an extract containing phytochemicals from pomegranates are disclosed. The processes generally comprise providing pomegranate solids, such as the pericarp, inner membrane and seeds; creating a mixture comprising the pomegranate solids in an aqueous solution; adding enzymes to the mixture in an amount sufficient to at least partially degrade the pomegranate solids; heating the mixture to a temperature that permits the maximum rate of catalysis of the enzyme; maintaining the temperature of the heated mixture for a time sufficient to allow at least partial degradation of the pomegranate solids; and removing residual insoluble solid materials from the mixture. Compositions containing the extract may be used as a food product, beverage, pharmaceutical preparations, nutritional supplements, vitamin supplements, food additives, and food supplements. The composition may also be used for preventing or ameliorating disease conditions by administering an effective amount of the composition to a subject in need thereof.

FIELD OF THE INVENTION

The present invention relates generally to pomegranate extracts, andmore particularly, to methods for obtaining and using extracts frompomegranate solids and compositions comprising pomegranate extracts.

BACKGROUND OF THE INVENTION

It is well-known that fruits and vegetables are an essential part of ahealthy diet. Chief, among the reasons, is that fruits and vegetablesare rich sources of important phytochemicals, which provide essentialnutrients and enhance the body's ability to prevent and fight disease.There is a multitude of phytochemicals, in unique combinations, indifferent fruits and vegetables, and each functions differently in thebody: as anti-oxidants, as anti-allergenic, as anti-carcinogenic, asanti-inflammatory, as anti-viral, and/or anti-proliferative.

The pomegranate has recently been acclaimed for its health benefits andfor its disease-fighting antioxidant potential. Antioxidants areimportant because they are believed to protect the body against freeradicals, the harmful molecules that can cause heart disease, prematureaging, Alzheimer's disease, blindness, and a variety of cancers.

Studies have shown that pomegranate juice has more polyphenolantioxidants than any other drink, such as red wine, green tea,blueberry juice, cranberry juice and orange juice. Currently, the twocommon ways of consuming pomegranates are by eating the fleshy arils ofthe pomegranate and by drinking the juice obtained from the arils.

There are many kinds of antioxidants, some produced by the body andothers derived from the foods we eat. When the body's naturalantioxidant defenses are lowered, or greater amounts of free radicalsare being produced, the body becomes more dependent upon food sources ofantioxidants.

BRIEF SUMMARY OF THE PREFERRED EMBODIMENTS

Methods are provided for producing an extract containing phytochemicalsfrom pomegranate solids. The pomegranate solids are any one or more ofthe group consisting of the pericarp, inner membrane and seeds. Theextract produced differs from commercially-available pomegranate juicein that the extract is substantially derived from the pomegranatesolids, whereas pomegranate juice is substantially derived from thesweet, fleshy arils.

In one preferred embodiment, the method includes the following steps.Any one or a combination of the pericarp, inner membrane and seeds areselected and a mixture is formed comprising the pomegranate solids andan aqueous solution. The mixture is then heated to about 60° F. to 210°F., preferably of about 85° F. to 185° F. and optimally of about 110° F.to 160° F. Enzymes are added to the mixture in an amount sufficient toat least partially degrade the pomegranate solids and liberatephytochemicals from the plant tissues and/or cells. Once liberated, thephytochemicals may react and/or polymerize to create new phytochemicalcompounds or reaction products. The residual insoluble solid materialsare removed from the mixture to provide an extract containingphytochemicals.

In another preferred embodiment, extracts containing phytochemicals froma pomegranate are provided. Such extracts are characterized by asignificantly higher total polyphenol content, particularly of the highmolecular weight polyphenol (e.g., punicalagin), than is found inpomegranate juice. Such extracts may be obtained from the methodsdisclosed herein.

In a further preferred embodiment, food products and beverages areprovided comprising the extract containing phytochemicals from apomegranate.

In yet a further preferred embodiment, compositions comprising theextract containing phytochemicals from a pomegranate are provided. Suchcompositions may be in form of tablets, suspensions, implants,solutions, emulsions, capsules, powders, syrups, liquid compositions,ointments, lotions, creams, pastes, and gels. Such compositions may alsobe in form of pharmaceutical preparations, nutritional supplements,vitamin supplements, food additives, and food supplements.

In a further preferred embodiment, compositions containing the extractand the pomegranate juice are provided. The combination of the extractand pomegranate juice not only produces a composition having a highertotal polyphenol content, as compared to the pomegranate juice alone,but it also provides the broad spectrum of the different polyphenolswhich predominate the pomegranate juice and extract.

In another preferred embodiment, methods are provided for preventing orameliorating disease conditions in a subject by administering to thesubject an effective amount of the composition suitable for use as apharmaceutical or nutritional preparation. Such disease conditionsinclude polyphenol-mediated diseases and cancer. Examples ofpolyphenol-mediated diseases include circulatory disorders such ashypertension and coronary artery disease, erectile dysfunction, lungdisorders such as asthma, cancers of various types, inflammatoryconditions, certain liver conditions, diabetes, mood disorders, eyedisorders such as cataracts, weak eyesight due to aging, maculardegeneration, and other age-related disorders, such as Alzheimer'sdisease and dementia.

In yet another preferred embodiment, methods are provided for modulatingthe growth and progression of cancerous cells, the methods comprisingselecting a subject having cancerous cell growth and administering tothe subject an effective amount of the composition containing theextract.

In yet a further preferred embodiment, methods are provided forpreventing or slowing increases in the Prostate Specific Antigen (PSA)levels in a subject having prostate cancer. The method comprisesselecting a subject having prostate cancer and administering to thesubject an effective amount of the composition containing the extract.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating preferredembodiments of the present invention, are given by way of illustrationand not limitation. Many changes and modifications within the scope ofthe present invention may be made without departing from the spiritthereof, and the invention includes all such modifications andequivalents thereof.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “phytochemicals” refers collectively tocompounds which are naturally-occurring in the pomegranate and toreaction products and metabolites of these compounds, which areconsidered to have a beneficial effect on the human health. Examples ofsuch phytochemicals include, but are not limited to polyphenols,estrogens and phytoestrogens.

As used herein, the term “polyphenols” refers generally to a family ofnaturally-occurring compounds in the pomegranate and includes phenolsand polyphenols. Phenols are a class of chemical compounds consisting ofa single phenol unit in their structure. Although similar to alcohols,phenols have unique properties including relatively higher acidities dueto the aromatic ring tightly coupled to the oxygen and a relativelyloose bond between the oxygen and the hydrogen. Examples of phenoliccompounds within this group include ellagic acid and gallic acid.Polyphenols are a group of compounds, characterized by the presence ofmore than one phenolic group. Polyphenols include tannins (e.g.,ellagitannins and gallotannins), flavonoids (e.g., anthocyanins andisoflavones) and stilbenes (e.g., resveratrol).

As used herein, the term “pomegranate juice” refers to the juice that issubstantially obtained from the arils of the pomegranate.

As used herein, the term “pomegranate solids” refers to any one or acombination of the pericarp, the inner membrane and seeds of apomegranate.

It has been surprisingly discovered that extracts obtained from thepomegranate solids, in accordance with the methods disclosed herein,have a substantially higher total polyphenol content than is found inthe juice from the pomegranate arils. This is particularly true withrespect to the higher molecular weight polyphenols and, in particular,punicalagin.

Punicalagin is a powerful antioxidant, protecting cardiovascularfunction and accurate cellular replication. Thus, punicalagin isresponsible, in part, for the high antioxidant activity of the extract.While the antioxidant and other beneficial health effects of the extractare due to the presence of polyphenols, the presence of otherphytochemical compounds in the extract, or the synergistic effect ofthese phytochemicals, may also be responsible for the anti-oxidant andother beneficial health effects of the extract.

In addition to punicalagin, other high molecular weight polyphenols havebeen characterized in the extract of pomegranate solids. These highmolecular weight polyphenols include ellagitannin and other hydrolysabletannins, such as punicacortein A, punicalin, pedunculagin, andgallotanin dimers and trimers.

Moreover, a large number of anthocyanins have been characterized in theextract of the pomegranate solids. Examples of the anthocyanins includepelargonidin 3-glucoside, cyaniding 3-glucoside, delphinidin3-glucoside, pelargonidin 3,5-diglucoside, cyaniding 3,5-diglucoside,and delphinidin 3,5-diglucoside. Although these anthocyanins have beencharacterized in both the pomegranate juice and the extract, these lowermolecular weight polyphenols comprise a higher proportion of the totalpolyphenol content in pomegranate juice (approximately 50%) than in theextract.

Accordingly, methods are provided for producing an extract containingphytochemicals from pomegranate solids. The extract produced from themethods disclosed herein differ from the commercially-availablepomegranate juice in that the extract is substantially derived from thepomegranate solids, whereas pomegranate juice is substantially derivedfrom the sweet, fleshy arils that surround the pomegranate seed. Theextract is characterized as containing polyphenols and, particularly,high molecular weight polyphenols, such as punicalagin.

In one preferred embodiment, the method comprises providing any one or acombination of pomegranate solids selected from the group consisting ofthe pericarp, inner membrane and seeds and creating a mixture comprisingthe pomegranate solids in an aqueous solution. In a preferredembodiment, the mixture of the pomegranate solids is created by addingwater in an amount that is about 20-80% w/v, and more preferably about50% w/v, of the pomegranate solids. The mixture is preferably crushed ormilled to create a rough grind of pomegranate solids dispersed in theaqueous solution.

The mixture is then heated to a temperature of about 60° F. to 210° F.,preferably of about 85° F. to 185° F. and optimally of about 110° F. to160° F. The temperature to which the mixture is heated depends upon theselection of enzymes, or combination of enzymes, that is added to themixture. Preferably, the mixture is heated to a temperature that permitsthe maximum catalysis of the enzyme or combination of enzymes.

Alternatively, enzymes may be added before the mixture is heated. Thus,the order of the steps of heating the mixture and adding the enzymes isnot critical, so long as the mixture is heated to a temperature thatpermits the enzymes to at least partially degrade the pomegranate solidsand liberate phytochemicals from the plant tissues and/or cells. Onceliberated, the phytochemicals may react and/or polymerize to create newphytochemical compounds or reaction products.

Enzymes suitable for use in accordance with this embodiment includethose which are capable of at least partially degrading the plant tissueor cells to liberate the phytochemicals from the pomegranate solids.Such enzymes include any one or a combination of pectinase, cellulase,hemicellulase, amylase, arabanase, and other hydrolyzing enzymes, toname a few. The enzymes added to the mixture may be naturally-occurringor synthetic. They may be derived from any one or a combination ofsources, such as animal, plant, fungal, and bacterial sources. Theamount of the enzyme or combination of enzymes added to the mixturedepends on the temperature of the mixture and the amount of pomegranatesolids present in the mixture.

After enzymes are added, the mixture is maintained at a temperature fora time sufficient to allow at least partial degradation of thepomegranate solids. The temperature and length of time required dependson the type of enzymes added to the mixture, the rate of enzymecatalysis and the amount of the pomegranate solids contained in themixture.

Thus, in one preferred embodiment, a combination of pectinase, cellulaseand hemicellulase enzymes are added to the mixture, which is heated to atemperature of about 60° F. to 210° F., preferably about 110° F. to 160°F., and optimally of about 120° F. The mixture is maintained at thesetemperature, preferably with agitation or stirring, for about 45-195minutes, preferably for about 45-75 minutes, and optimally for about 60minutes.

After the enzymes have at least partially degraded the pomegranatesolids, the residual insoluble solid materials are removed from themixture. Optionally, a clarification agent, such as bentonite, may beadded before the step of removing the residual insoluble materials fromthe mixture. The removal of residual insoluble materials from themixture may be accomplished by filtration, centrifugation,chromatographic techniques, and other techniques. Filtration techniquessuitable for the practice of the methods disclosed herein includemicro-filtration at a molecular weight cut-off of at least 1,000 Da,preferably of about 4,500 Da, and optimally of about 5,500 Da.

The resulting liquid extract may be concentrated in an evaporator undervacuum to about 50-90 Brix (Bx), preferably to about 60-80 Bx, andoptimally to about 70 Bx, and pasteurized at a temperature and for alength of time sufficient to kill microorganisms that could causedisease, spoilage or undesired fermentation. In one preferredembodiment, the extract may be pasteurized at a temperature of about140° F.-280° F., preferably of about 195° F.-240° F., and optimally ofabout 205° F. The pasteurization may also denature the remaining enzymesthat were added to the mixture.

In another preferred embodiment, extracts containing phytochemicals froma pomegranate are provided. Such extracts are characterized by asignificantly higher total polyphenol content, particularly of the highmolecular weight polyphenol (e.g., punicalagin), than is found inpomegranate juice. Such extracts may be obtained from the methodsdisclosed herein. In a further preferred embodiment, extracts containingphytochemicals, polyphenols, punicalagin, punicalin, ellagic acid, andmetabolite thereof are provided.

In yet another preferred embodiment, food products and beverages areprovided comprising the extract containing phytochemicals from apomegranate. For example, due to the significantly higher totalpolyphenol content in the extract, an 8 oz sports beverage containing0.33 oz of the extract may be formulated to deliver the same totalpolyphenols as a 20 oz single-strength pomegranate juice. The polyphenolcontent of pomegranate juice is approximately about 1 to 2.25 mg/mL andthe amount of polyphenols present in 20 oz of juice is approximately 567to 1,256 mg. In contrast, the extract may contain a polyphenol contentof about 60 to 120 mg/mL, depending on the method employed. Thus only0.33 oz of the 70 Bx extract would be needed to provide the equivalentamount of polyphenols in 20 oz of the juice.

In a further preferred embodiment, compositions comprising the extractcontaining phytochemicals from a pomegranate are provided. Thecompositions may be formulated in the form of tablets, suspensions,implants, solutions, emulsions, capsules, powders, syrups, liquidcompositions, ointments, lotions, creams, pastes, gels, and the like.

The compositions may also be prepared in forms suitable for use aspharmaceutical preparations, nutritional supplements, vitaminsupplements, food supplements, and food additives. As such, thecompositions may optionally include a suitable carrier or excipient.

Suitable carriers or excipients are inert ingredients and include, byway of example, fillers, e.g. sugars such as lactose, glucose orsucrose, sugar alcohols such as mannitol, sorbitol or xylitol, starchsuch as wheat, corn or potato starch, modified starch or sodium starchglycolate, lubricants such as talc, magnesium stearate, calciumstearate, colloidal silica or stearic acid, and binders such aspolyvinylpyrrolidone, cellulose derivatives, carboxymethyl cellulose,hydroxylpropyl cellulose, hydroxypropylmethyl cellulose, methylcellulose or gelatin. Conventional procedures for preparing suchcompositions in appropriate dosage forms of the extract may be utilized.Such compositions may be administered orally or parenterally employingliquid form preparations containing the extract.

The compositions may be administered orally, in appropriate dosage unitsof the extract in a pharmaceutically acceptable carrier or excipient.Thus, the compositions may be formulated into solid or liquidpreparations, such as capsules, pills, tablets, powders, solutions,suspension, or emulsions and may be prepared according to methods knownin the art for the manufacture of such compositions. The solid unitdosage forms may be in form of a hard or soft shelled gelatin capsulecontaining the extract and a suitable carrier or excipient.

The composition may also be administered parenterally as injectabledosages in a physiologically acceptable carrier. Parenteraladministration may be subcutaneous, intravenous, intramuscular, orinterperitoneally.

The effective amount of a composition is the amount or dosage unit ofthe extract sufficient to achieve the intended beneficial healthresults. Accordingly, the effective amount of the composition to beadministered depends on considerations such as the dosage unit employed,the mode of administration, the period of treatment, the age, sex andweight of the person treated and the nature and extent of the conditiontreated. The effective amount can readily be determined based uponstandard techniques known to evaluate whether the intended effect of thecomposition has been achieved, by standard toxicity tests and bystandard pharmacological assays.

In a further preferred embodiment, compositions containing the extractand the pomegranate juice are provided. The combination of the extractand pomegranate juice not only produces a composition having a highertotal polyphenol content, as compared to the pomegranate juice alone,but it also provides the broad spectrum of the different polyphenolswhich predominate the pomegranate juice and extract, for example thelower molecular weight polyphenols (e.g., anthocyanins) which is presentin greater quantities in the pomegranate juice and the higher molecularweight polyphenols (e.g., punicalagin, punicalin, ellagic acidglycosides, ellagic acid polyphenols, ellagitannin and otherhydrolysable tannins, such as punicacortein A, punicalin, pedunculagin,and gallotanin dimmers and trimers).

In yet a further preferred embodiment, methods are provided forameliorating disease conditions in a subject by administering to thesubject an effective amount of the composition suitable for use as apharmaceutical or nutritional preparation. Such disease conditionsinclude polyphenol-mediated diseases and cancer.

Polyphenols and countless other phytochemicals in the extract arenecessary for the various organs and tissues and for the properfunctioning of the human body. Accordingly, many disease conditions maybe prevented or ameliorated by the administration of polyphenols topatients with polyphenol-mediated diseases. These polyphenol-mediateddiseases include circulatory disorders such as hypertension and coronaryartery disease, erectile dysfunction, lung disorders such as asthma,cancers of various types, inflammatory conditions, certain liverconditions, diabetes, mood disorders, eye disorders such as cataracts,weak eyesight due to aging, macular degeneration, and other age-relateddisorders, such as Alzheimer's disease and dementia.

Thus, in one preferred embodiment, methods are provided for formulatinga composition suitable for use as a pharmaceutical or nutritionalpreparation for improving the health of a subject comprising obtainingan extract containing phytochemicals from a pomegranate and admixing aneffective amount of the extract with a suitable carrier or excipient.

In another preferred embodiment, methods are provided for treating apolyphenol-mediated condition in a subject comprising selecting asubject having a polyphenol-mediated condition and administering to thesubject an effective amount of the composition comprising the extract.

In yet another preferred embodiment, methods are provided for modulatingthe growth and progression of cancerous cells, the methods comprisingselecting a subject having cancerous cell growth and administering tothe subject an effective amount of the composition containing theextract.

In yet a further embodiment, methods are provided for preventing orslowing increases in the Prostate Specific Antigen (PSA) levels in asubject having prostate cancer. The method comprises selecting a subjecthaving prostate cancer and administering to the subject an effectiveamount of the composition containing the extract.

Prostate cancer is the most commonly detected cancer in men in the U.S.,affecting approximately 1 out of every 6 men. It is the second leadingcause of cancer death among men in the U.S. Laboratory testing canassist with screening, diagnosis, staging, prognosis, detection ofresidual or recurrent disease, and therapeutic monitoring. The primarytest used for these purposes is a PSA test.

The PSA test was approved by the FDA in 1986 to help detect prostatecancer. A number of prostate problems can be identified by testing andmonitoring the levels of PSA circulating in the bloodstream. The levelof PSA in the bloodstream may be elevated by an process that leads to anincrease in the number of cells making PSA or to a breakdown of thenormal barriers in the prostate that prevent much PSA from getting intothe bloodstream.

Increases in levels of PSA in the blood following treatment forlocalized prostate cancer with surgery or radiation often indicates thepresence of residual cancer and the eventual development of metastaticcancer. Moreover, PSA doubling times are correlated with diagnostictumor stage and grade

The following examples further illustrate the embodiments disclosedherein. These examples are provided only for purpose of illustrating thepreferred embodiments of the invention and do not limit the invention inany manner.

EXAMPLE 1 Production of Liquid Extract from Pomegranate Solids

The starting material for the production of the extract is thepomegranate solids, which generally comprise the pericarp, the innermembrane and seeds of the pomegranate. The pomegranate solids wereobtained and collected after the primary juice from the arils had beensubstantially expelled or otherwise removed from the pomegranate bypressing, crushing, or other methods known to the art for extractingpomegranate juice.

The pomegranate solids were then transferred to three Reitz Mills with⅜-inch screens. The material was milled to a fine puree and heated toapproximately 125° F. This step, coupled with the following enzymeaddition, assisted in breaking down the colloidal structure of theremaining pomegranate solids, thereby releasing the remaining solublesolids.

The mixture was heated to a temperature of about 125° F. for two hours.Three enzymes were added to the mixture: pectinase (Rohapect® DA6L),cellulase/pectinase (Rohapect® CL), and hemi-cellulase/pectinase(Rohapect® B1L). These enzymes were used to liberate the remainingpomegranate soluble solids, such as sugars, minerals, anthocyanins, andremaining polyphenols.

The mixture was then pumped from the extraction plant to the primaryprocessing plant where it was held in the mash treatment tanks forapproximately one hour. After one hour, 50-100 pounds of bentonite in a125 galloon water slurry, per 8,000 gallons of the mixture, was addedfor protein removal. The treated mixture was then passed through aWestphalia 755 Decanter for removal of solids. The residual insolublematerial was discharged as waste.

The remaining liquid extract was processed in a Schmidt evaporator. Inthis step, the extract was stripped and rectified. In addition, theliquid extract was pre-concentrated and then pasteurized to 205° F. for45 seconds. The liquid extract then exited the evaporator and wasfiltered on Koch Micro-Filtration membranes at a 4,500 Da molecularweight cut-off for liquid extract soluble solids.

The liquid extract then re-entered the evaporator for finalconcentration. Initial heat on this step was about 185-195° F. At about70 Bx, the liquid extract was cooled to less than about 45° F. andpumped to the concentrate batching room where it was blended andstandardized.

EXAMPLE 2 Comparison of Polyphenol Content in Extracts of PomegranateSolids and in Pomegranate Juice

The concentrations of punicalagin, punicalin, ellagic acid glycosides,and ellagic acid polyphenols in the pomegranate juice and thepomegranate extract were analyzed and compared in a University study.

All samples (50 mL injection volume) were filtered (0.22 mm) andanalyzed on a Novapak (Waters Corp.) C-18 column, 150×3.9 mm i.d., 5 mm.The mobile phase, solvent A (2% CH₃COOH/H₂O) and solvent B (2% aqueousCH₃COOH/CH₃OH) was used under linear gradient conditions starting with99% solvent A in solvent B to 40% solvent A in solvent B over 40minutes, hold time, 5 minutes with a flow rate of 1 mL/min. Allcompounds were detected at 254 nm, and at 378 nm (punicalagins) and 366(ellagic acid) for quantification.

Table 1 shows a side-by-side comparison of the concentration of thepolyphenols punicalagins, punicalin, ellagic acid glycosides, andellagic acid in the pomegranate extract and the pomegranate juice.

TABLE 1 Pomegranate Extract Pomegranate Juice ConcentrationConcentration Compound Name (mg/ml) (mg/ml) Punicalagin (β- 4.79 0.02isomer) Punicalagin (α- 21.80 0.15 isomer) Punicalin 3.62 NA EllagicAcid 19.65 0.33 Glycosides Ellagic Acid 18 0.74 Total: 67.86 1.24

Although other polyphenols are present in both the pomegranate extractand juice, and this example highlights the unexpected and surprisingresults in that significantly higher concentrations of polyphenols,particularly of punicalagin, are present in the pomegranate extract thanin the pomegranate juice. Table 1 shows a total punicalagin (for both α-and β-isomers) concentration for the pomegranate extract that is over26-fold greater than for the pomegranate juice.

EXAMPLE 3 Effects of Pomegranate Juice in Men with Rising PSA FollowingSurgery or Radiation for Prostrate Cancer

The positive and significant beneficial effects of pomegranate juice onProstrate Specific Antigen (PSA) parameters have been demonstrated in aclinical trial in patients with recurrent prostrate cancer, coupled withcorresponding laboratory effects on prostrate cancer in in vitro cellgrowth and apoptosis.

To determine the clinical effects of pomegranate juice on patients withprostate cancer, a clinical trial was performed. A two-year, singlecenter, phase II, Simon two stage clinical trial for men with rising PSAafter surgery or radiotherapy was designed based on a 20% response rate,an alpha of 5%, and 90% power. Eligible patients had a detectable PSAgreater than 0.2 ng/ml and less than 5 ng/ml, and a Gleason score of 7or less. Serial PSA measurements determined a baseline PSA doublingtime.

Patients were treated with 8 oz of pomegranate juice by mouth daily(Wonderful variety, equivalent to 1.5 mmol of total polyphenols per day)until disease progression. Clinical endpoints included safety, effect onserum PSA, and exploratory laboratory studies. Patients were followed in3-month intervals for serum PSA, and blood and urine were collected forlaboratory studies.

The study was fully accrued to 48 participants in two stages afterefficacy criteria were met. There were no serious adverse eventsreported and the treatment was well tolerated. No patients developedmetastatic disease on study. Mean PSA doubling time significantlyincreased with treatment, from a mean of 14 to 26 months (p<0.048). Theslope of the mean log PSA decreased from 0.08 to 0.04 on treatment(p<0.019). In vitro assays using pre- and post-treatment patient serumon the growth of LNCaP showed decreased cell proliferation and increasedapoptosis (p<0.07). Pomegranate polyphenols were detected in the urineof all participants by liquid chromatography mass spectrometry (LC-MS).

EXAMPLE 4 Preventing or Slowing Increases in the PSA Levels of PatientsWith Prostate Cancer

While both pomegranate juice and pomegranate solid extract containvarious types of the anti-oxidant polyphenols, pomegranate solid extractcontains a higher total polyphenol content than the pomegranate juice.Accordingly, to the extent that the administration of 8 oz ofpomegranate juice to patients with prostate cancer has been demonstratedto increase the PSA doubling time in patients with prostate cancer, theadministration of pomegranate solid extract also achieves at least thesame, and preferably an improved, effect.

Accordingly, methods and compositions are provided for preventing orslowing increases in the Prostate Specific Antigen (PSA) levels in apatient. The methods comprise selecting a subject having prostate cancerand administering to the subject an effective amount of a compositioncontaining the extract containing phytochemicals from a pomegranatesolid.

In preferred one embodiment, the composition may be in form of a liquidcomprising the extract and pomegranate juice. The total polyphenolcontent provided by the liquid may be varied by the changing the amountof the pomegranate extract and pomegranate juice contained in theliquid. Table 2 provides examples of the formulations of the liquidcomposition and the total polyphenol content in the formulationsrelative to the total polyphenol content in standard pomegranate juice.

TABLE 2 Extract/Pomegranate Pomegranate Pomegranate Juice Liquid ExtractJuice Composition (oz) (oz) Formulation 1 0.13 8 2× polyphenol contentFormulation 2 0.26 8 3× polyphenol content Formulation 3 0.39 8 4×polyphenol content Formulation 4 0.52 8 5× polyphenol content

For purposes of this embodiment, the effective amount of the extractthat is administered to the patient is at least 0.13 oz (or anequivalent unit or measurement) of the extract administered daily,whether the extract is provided alone or in a composition. Because theadministration of 8 oz of pomegranate juice was found to be effective inslowing the rising PSA levels in patients with prostate cancer, theadministration of at least 0.13 oz of the pomegranate solid extract isbelieved to achieve the same, if not improved, results. The dosage ofthe extract may be increased by administering a greater dosage orincreasing the frequency at which the extract is administered.

In addition to the liquid compositions containing the extract, theextract may also be administered in a solid form, such as pharmaceuticalor nutritional preparation that comprise the extract and apharmaceutically acceptable carrier or excipient.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims

1. A method of modulating growth and progression of cancerous cells, themethod comprising: selecting a subject having cancerous cell growth;administering to said subject a composition comprising an effectiveamount of a pomegranate extract, whereby said pomegranate extract isproduced by a process comprising: providing one or more pomegranatesolids selected from the group consisting of a pericarp, inner membraneand seeds; creating a mixture comprising said one or more pomegranatesolids in an aqueous solution; adding hydrolyzing enzymes to saidmixture in an amount sufficient to at least partially degrade said oneor more pomegranate solids; heating said mixture to a temperature thatpermits enzyme catalysis of said pomegranate solids; maintaining saidmixture at a temperature and time sufficient to allow at least partialdegradation of the pomegranate solids; and removing residual insolublesolid materials from said mixture to provide an extract, wherein saidextract contains polyphenols.
 2. A method of modulating growth andprogression of cancerous cells comprising: selecting a subject havingcancerous cell growth; administering to said subject a compositioncomprising an effective amount of a pomegranate extract and pomegranatejuice, whereby said pomegranate extract is produced by a processcomprising: providing one or more pomegranate solids; creating a mixturecomprising said one or more pomegranate solids in an aqueous solution;adding hydrolyzing enzymes to said mixture in an amount sufficient to atleast partially degrade said one or more pomegranate solids; heatingsaid mixture to a temperature that permits enzyme catalysis of saidpomegranate solids; maintaining said mixture at a temperature and timesufficient to allow at least partial degradation of said one or morepomegranate solids; and removing residual insoluble solid materials fromsaid mixture to provide an extract, wherein said extract containspolyphenols.
 3. The method of claim 2 wherein said pomegranate solid isselected from the group consisting of pericarp, inner membrane and seedsfrom a pomegranate fruit.
 4. The method of claim 3 wherein saidpomegranate solid comprises the remainder of a whole pomegranate fruitfollowing extraction of substantially all liquid from arils of saidwhole pomegranate fruit.
 5. A method of increasing Prostate SpecificAntigen doubling time in a subject having prostate cancer comprising:selecting a subject having prostate cancer; administering to saidsubject a composition comprising an effective amount of a pomegranateextract, whereby said pomegranate extract is produced by a processcomprising: obtaining one or more pomegranate solids; creating a mixturecomprising said one or more pomegranate solids in an aqueous solution;adding hydrolyzing enzymes to said mixture in an amount sufficient to atleast partially degrade said one or more pomegranate solids; heatingsaid mixture to a temperature that permits enzyme catalysis of said oneor more pomegranate solids; maintaining said mixture at a temperatureand time sufficient to allow at least partial degradation of said one ormore pomegranate solids; and removing residual insoluble solid materialsfrom said mixture to provide an extract, wherein said extract containspolyphenols.
 6. The method of claim 5 wherein said pomegranate solid isselected from the group consisting of pericarp, inner membrane and seedsfrom a pomegranate fruit.
 7. The method of claim 5 wherein saidpomegranate solid comprises the remainder of a whole pomegranate fruitfollowing extraction of substantially all liquid from arils of saidwhole pomegranate fruit.
 8. A method of increasing Prostate SpecificAntigen doubling time in a subject having prostate cancer, the methodcomprising: selecting a subject having prostate cancer; administering tosaid subject a composition comprising an effective amount of apomegranate extract and pomegranate juice, whereby said pomegranateextract is produced by a process comprising of providing one or morepomegranate solids; creating a mixture comprising said one or morepomegranate solids in an aqueous solution; adding hydrolyzing enzymes tosaid mixture in an amount sufficient to at least partially degrade saidpomegranate solids; heating said mixture to a temperature that permitsenzyme catalysis of said one or more pomegranate solids; maintainingsaid mixture at a temperature and time sufficient to allow at leastpartial degradation of said one or more pomegranate solids; and removingresidual insoluble solid materials from said mixture to provide anextract, wherein said extract contains polyphenols.
 9. The method ofclaim 8 wherein said pomegranate solid is selected from the groupconsisting of pericarp, inner membrane and seeds from a pomegranatefruit.
 10. The method of claim 8 wherein said pomegranate solidcomprises the remainder of a whole pomegranate fruit followingextraction of substantially all liquid from arils of said wholepomegranate fruit.